Human phospholipase activating protein and methods for diagnosis of rheumatoid arthritis

ABSTRACT

The invention provides methods for detecting elevated levels of phospholipase A 2  activating protein in persons suspected of having rheumatiod arthritis to thereby indicate the presence of rheumatoid arthritis in the person comprising the steps of providing a sample of body fluid or tissue from said person; contacting the sample with an antibody specific for phospholipase A 2  activating protein such that the antibody binds with phospholipase A 2  activating protein in the sample; detecting the antibody thereby indicating the presence of phospholipase A 2  activating protein, whereby elevated levels of phospholipase A 2  activating protein in the sample as compared with levels found in persons not having rheumatoid arthritis indicates the presence of rheumatoid arthritis in the person. Kits and reagents for detecting rheumatoid arthritis are also provided.

This is a division of application Ser. No. 07/626,589, filed Dec. 6,1990, now U.S. Pat. No. 5,294,698.

FIELD OF THE INVENTION

The present invention relates to the field of methods for diagnosingrheumatoid arthritis. More particularly the present invention relates tomethods for diagnosing rheumatoid arthritis using immunoassays.

BACKGROUND OF THE INVENTION

Rheumatoid arthritis is the best known form of arthritic disease,affecting millions of patients worldwide. It is characterized by aprogressive inflammation of joints and internal organs byimmunocompetent cells and destructin of articular cartilage resulting inprogressive morbidity and death. Prostaglandins and related eicosanoidsare thought to be important mediators of these immune and inflammatoryresponses. Increased quantities of eicosanoids are produced byrheumatoid synovium in both organ and cell culture and are found inelevated levels in rheumatoid synovial fluid and from peripheral bloodcells of affected patients. Clinical evidence suggests that earlydiagnosis and intervention with cytotoxic and immunomodulatory agents isessential to altering the course of the disease.

Phospholipase A₂ (PLA₂) is a lipolytic enzyme which hydrolyzes the2-acyl fatty acid ester of glycerophospholipids, thus releasingarachidonic acid. Arachidonic acid has been shown to be converted into anumber of biologically active compounds known as eicosanoids. PLA₂activity has been shown to be ubiquitous and to reside in severaldifferent gene products. Both membrane bound and soluble forms of theenzyme have been described. Extracellular secretion of soluble PLA₂ wasfirst described in 1980. Circulating PLA₂ was implicated in endotoxinshock and has since been implicated in acute and chronic inflammatoryreactions.

Currently rheumatoid arthritis is diagnosed on the basis of thepatient's physical presentation and a limited number of laboratorytests. Most of these tests indicate a generalized inflammatory state anddiagnosis is only reinforced on the basis of cumulative results. Of thelaboratory tests designed to be more specific, the presence ofrheumatoid factor has been shown to correlate with approximately 75% ofbona-fide rheumatoid arthritis cases and attempts have been made toestablish rheumatoid factor as a distinguishing criteria from otherinflammatory diseases. Incorrect diagnoses of rheumatoid disease, basedupon rheumatoid factor, however, do occur with hepatitis sarcoidosis andinfections, as well as other inflammatory joint diseases such as Reiterssyndrome. In addition at least 5% of disease free individuals over age65 test positive for the presence of rhematoid factor. Further, elevatedor depressed levels do not correspond well with flare-ups or remissionsin rheumatoid disease. Clearly, rheumatoid factor is neither sensitivenor specific for the diagnosis or prognosis of rheumatoid arthritis andother more sensitive and specific methods for diagnosing and followingthe fourse of the disease are needed.

It is thus an object of the invention to provide such methods. It isalso an object of the invention to provide test kits and reagents fordiagnosing or following the course of rheumatoid arthritis in personshaving the disease. It is a further object of the invention to providenucleic acid sequences, polypeptides and antibodies useful in diagnosingor following the course of rheumatoid arthritis. It is yet anotherobject of the invention to provide antisense oligonucleotides and othersubstances useful in the treatment of rheumatoid arthritis.

SUMMARY OF THE INVENTION

The present invention provides a novel human phospholipase A₂ activatingprotein (PLAP). The novel phospholipase A₂ activating protein of theinvention was isolated from synovial fluid of patients presenting withbona fide rheumatoid arthritis. The invention also provides methods,kits, and reagents for diagnosing and following the bourse of rheumatoidarthritis. The invention further provides a nucleic acid sequence codingfor PLAP.

The correlation of elevated levels of this protein in rheumatoidarthritic patients over levels found in the synovial fluid of patientswith osteoarthritis (degenerative arthritis), the prototypicalinflammaotory arthritic disease, is greater than 87%. In addition levelsof the protein are likely to correspond to remission or flare-ups of thedisease. In contrast patients with other forms of inflammatory jointdisease such as Reiter's syndrome, and pseudogout or ankylosingspondylitis fail to show elevated PLAP levels. Assays for PLAP arelikely to be unaffected by hepatitis, sarcoidosis, or most infections.Thus, levels of PLAP in synovial fluid and possibly serum can be usedfor the diagnosis and monitoring of rheumatoid arthritis without thosedifficulties and uncertainties associated with rheumatoid factor.

This invention is more particularly pointed out in the appended claimsand is described in its preferred embodiments in the followingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of PLAP fragments tested for PLA₂ stimulatingactivity.

FIGS. 2a-2c show the sequence of PLAP cDNA. The predicted amino acidsequence of the largest open reading frame is also shown. The predictedamino acid sequence begins a 1 (underlined ATG) The region having thegreatest homology with melittin is highlighted and underlined. Thecomplimentary antisense DNA used in Tables 8, 9, and 10 was synthesizedusing the region encoded between the two arrows which begins at thecodon after ATG, methionine which is 1.

FIG. 3 shows a graph of the PLA₂ activity of fractions of PLAP collectedfrom a anti-melittin affinity chromatography column during purificationof PLAP from human synovial fluid.

FIG. 4 shows a graph of the PLA₂ activity of fractions of PLAP collectedfrom a size exclusion chromatography column during purification of PLAPfrom human synovial fluid.

FIG. 5 shows PLAP induced release of [³ H]-arachiconic acid metabolitesfrom human peripheral blood monocytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel human phospholipase A₂ activatingprotein (PLAP) useful in assays for diagnosing and following the courseof rheumatoid arthritis.

The invention also provides a method for detecting elevated levels ofphospholipase A₂ activating protein in persons suspected of havingrheumatoid arthritis to thereby indicate the presence of rheumatoidarthritis in the person comprising the steps of providing a sample ofbody fluid or tissue from said person; contacting said sample with anantibody specific for phospholipase A₂ activating protein such that saidantibody binds with phospholipase A₂ activating protein in the sample;and detecting the antibody thereby indicating the presence ofphospholipase A₂ activating protein, whereby elevated levels ofphospholipase A₂ activating protein in the sample as compared withlevels found in persons not having rheumatoid arthritis indicates thepresence of rheumatoid arthritis in the person.

Another embodiment of the invention provides substantially purifiednucleic acid sequences coding for the phospholipase A₂ activatingprotein. A nucleic acid sequence coding for phospholipase A₂ activatingprotein isolated from bovine cells is shown in FIG. 2. The predictedamino acid sequence of PLAP is also shown.

A further embodiment of the invention provides a method for purifyinghuman phospholipase A₂ activating protein from body fluid or tissuecomprising the steps of a) contacting the body fluid or tissue withanti-melittin or anti-phospholipase A₂ activating protein antibodiesbound to a solid support wherein phospholipase A₂ activating protein inthe body fluid or tissue binds with anti-melittin or anti-phospholipaseA₂ activating protein antibodies; b) eluting bound phospholipase A₂activating protein from said solid support; c) passing elutedphospholipase A₂ activating protein from step b through a size exclusioncolumn; and d) collecting purified phospholipase A₂ activating protein.A preferred solid support is silica. The eluting step is preferablyperformed with sodium acetate buffer.

Another embodiment of the invention provides a method of detectingphospholipase A₂ activating protein in a mammalian body fluid or tissuecomprising the steps of contacting a sample of the body fluid with anantibody specific for phospholipase A₂ activating protein whereby theantibody binds to phospholipase A₂ activating protein in the body fluidor tissue, and detecting the antibody thereby indicating the presence ofphospholipase A₂ activating protein in the body fluid or tissue. Thedetecting step may further comprise contacting bound antibody specificfor phospholipase A₂ activating protein with a detectably labeledantibody bindable with the bound antibody; and detecting the detectablelabel. In a preferred embodiment the detectable label is peroxidase andthe detecting step contacting the peroxidase with O-phenylenediaminesubstrate and determining optical density of the substrate product.

The invention also provides reagents and kits for detecting rheumatoidarthritis and phospholipase A₂ activating protein in mammalian bodyfluid or tissue. The reagents of the invention comprise comprising anantibody specific for phospholipase A₂ activating protein and a carrieror diluent. Kits of the invention comprise a solid surface having anantibody specific for phospholipase A₂ activating protein bound thereon,at least one reagent comprising an antibody specific for phospholipaseA₂ activating protein and a carrier or diluent; and means for detectingthe antibody.

The invention further provides a method of inhibiting the synthesis ofphospholipase A₂ activating protein in mammalian cells comprising thesteps of contacting a mammalian cell with antisense nucleic acidsequence coding for a phospholipase A₂ activating protein synthesisinhibiting sequence of phospholipase A₂ activating protein whereby saidantisense nucleic acid sequence inhibits synthesis of phospholipase A₂activating protein. The antisense sequence may be prepared as DNA orRNA.

A further embodiment of the invention provides a method of treatingmammalian, preferably human, rheumatoid arthritis comprisingadministering to a mammal a PLAP synthesis inhibiting amount of anantisense nucleic acid sequence coding for at least a portion of PLAP.

The destructive effects of rheumatoid arthritis on joints is associatedwith enhanced production of eicosanoids derived from arachidonic acid.Increased quantities of eicosanoids are produced by rheumatoid synoviumin both organ and cell culture and are found in elevated levels inrheumatoid synovial fluid and from peripheral blood cells of affectedpatients.

At the present time, persons having rheutamoid arthritis are treatedwith non-steroidal anti-inflammatory drugs which inhibit the productionof eicosanoids. The rate limiting step in eicosanoid biosynthesis is therelease of arachidonic acid from membrane phospholipids by phospholipaseenzymes, so that activation of arachidonic acid release by PLAP wouldeffectively increase eicosanoid synthesis by making available itsbiosynthetic precursor. PLAP thus appears to be an important mediator ofthe effects of rheumatoid arthritis.

Applicants have discovered that PLAP induces eicosanoid release andstimulation of joint inflammation. To demonstrate PLAP induction ofeicosanoid release, normal human peripheral blood monocytes preloadedwith labelled arachidonic acid were stimulated with PLAP. A signifigantrelease of arachidonic acid metabolites was observed. Radioimmunoassayfor specific metabolites of unlabelled cells showed prostaglandin E₂ asthe major metabolite released. PLAP was also shown to stimulate releaseof leukotriene B₄ and prostaglandin E₂.

PLAP stimulation of an inflammatory arthritogenic response was examinedby injection of purified PLAP into rabbit knee joints. PLAP was found tohave a dose-response relationship in mediating an inflammatoryarthropathy with cellular infiltration and joint destruction.

To determine the cellular source of PLAP found in human joints,metacarpophalngeal and knee joint specimens were obtained from patientswith rheumatoid arthritis or osteoarthritis and immunohistochemicalstaining with anti-PLAP antibodies performed. All 6 rheumatoid arthriticpatients tested displayed intense staining. No staining was observedwith the specimens obtained from osteoarthritic patients. Microscopicexamination revealed heavy staining of synovial monocytes, macrophages,and multinucleated giant cells with some additional staining of vascularsmooth muscle and endothelial cells. Similar results were observed inrheumatoid nodules. Osteoarthritic synovium revealed no PLAP stainingeven under microscopic examination. No staining was observed in eitherspecimen type with pre-immune antisera.

In order to quantitate the presence of PLAP as a function of thepresence and stage of rheumatiod arthritic disease ELISA assays wereperformed on a broad spectrum of synovial fluid samples drawn fromrheumatoid arthritic patients, normal healthy volunteers, and patientswith other form of arthritis or joint disease. Specimens from patientswith rheumatoid arthritis showed an average 4.3-fold increase in PLAPlevels over healthy synovial fluid or fluid from patients withosteoarthritis. Within the detection limits of the assay 87% ofrheumatoid arthritic patient synovial fluid showed elevated PLAP levels.Elevated levels of PLAP were detected in rheumatoid arthritic patientsand increased levels of the activating protein were associated withseverity of the disease (p<0.05).

Applicants have found that PLAP has a biochemical affinity for a subsetof PLA₂ activity selective for lipolysis of phosphatidylcholine,possibly like melittin preferring arachidonic acid as the sn-2 fattyacid. Indeed the specificity of PLAP is particularly relevant torheumatoid arthritis since phosphatidylcholine andlysophosphatidylcholine are the major phospholipids in inflammatorysynovium and synovial fluid aspirated from rheumatoid arthriticpatients.

The extensive tissue necrosis and hemorrage associated with injection of100,000 PLAP units is similar to that observed in other experimentalsystems with the injection of melittin (Kurihara Cell Tissue Res.(1986)). Since over 400,000 units of PLAP could be recovered from aslittle as 10 ml of synovial fluid from rheumatoid arthritic patients itis clear that pathophysiologic concentrations of PLAP may play a keyrole in mediating the disease.

Purified human PLAP has an apparent molecular mass of about 28,000 asdetermined by SDS gel electrophoresis. PLAP may be purified from humansynovial fluid or other human fluid or tissue containing the protein byaffinity chromatography using immobilized anti-mellitin antibodies orantibodies specific for PLAP, followed by high pressure liquidchromatography (HPLC) using a size exclusion gel, preferably a silicagel, or any other conventional method, or combination of methods forisolating proteins that results in substantially purified PLAP.Preferred methods for isolating PLAP from human synovial fluid aredescribed in detail in the Examples. PLAP is preferably isolated fromsynovial joint fluid of persons having rheumatoid arthritis, as thelevel of PLAP is higher in these persons. However, synovial fluid frompersons not having rheumatoid arthritis is also suitable, althoughlarger quantities of synovial fluid may be necessary due to thecomparatively lower amount of PLAP in the synovial fluid of persons nothaving rheumatoid arthritis. Methods for affinity chromatography, highpressure liquid chromatography and other methods useful for isolatingproteins may be found in the scientific literature and standard texts inthe field such as Scopes, R. K., Protein Purification, Principles andPractice, second edition, Springer-Verlag, New York, N.Y. 1987.

In addition to purification from natural sources, PLAP may also beobtained using recombinant DNA techniques. A nucleic acid sequencecoding for PLAP may be obtained from mammalian cells by amplification ofnucleic acid using nucleic acid primers having the sequence of thebeginning or end portions of PLAP (human or from other species) andpolymerase chain reaction procedures, followed by insertion of theamplified nucleic acid into a cloning vector such as lambda gt11, andsubsequently inserting the amplified sequence into an expression vectorand host cell, and screening for the sequence coding for PLAP usingantibodies specific for PLAP. A nucleic acid sequence coding for PLAPmay also be obtained by screening a DNA library (genomic or cDNA) fromthe cells of a mammal for the sequence, or for shorter sequences thattaken together code for the entire sequence of PLAP, as described inSambrook et al., Molecular Cloning, A Laboratory Manual, second edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Apreferred method for isolating a nucleic acid sequence coding for PLAPis described in the Examples. A preferred cell line for isolation ofPLAP is BC 3H1, a murine smooth muscle-like brain tumor cell lineobtainable from the American Type Culture Collection (accession numberCRL 1443). As an alternative to the BC3H1 cell line, other types ofmammalian cells and cell lines such as human cells and cell lines may beused for isolation of a nucleic acid sequence coding for PLAP. Nucleicacid coding for PLAP isolated from any source is within the scope of theinvention, and is suitable for the purposes of the invention asdescribed herein.

Fragments or subportions of PLAP having immunogenic activity or PLA₂stimulating activity are also within the scope of the invention.Fragments of PLAP having immunogenic activity includes fragments of PLAPof any size that are capable of serving as antigen for production ofantibodies specific for PLAP, whether or not conjugated to a carrierprotein. As used herein PLA₂ activating activity, PLA₂ stimulatingactivity and similar terms are intended to refer to increasing PLA₂activity to levels above the level of endogenous PLA₂ activity found incontrols in the PLA₂ activating assays described herein. Thus a PLA₂stimulating or activating portion of PLAP is one that stimulates PLA₂activity to levels above those found endogenously.

Fragments of PLAP include polypeptides or peptides having fewer aminoacids than PLAP. Preferred fragments of PLAP are from about six to about30 amino acids in length, more preferably about 26 amino acids inlength. Fragments of PLAP are preferably selected such that the aminoacid sequence of the fragment contains at least a part of the sequencehomologous with melittin. Melittin is a low molecular weight peptidecontaining 26 amino acids found in bee venom that has phospholipaseactivating activity.

The amino acid sequence of PLAP shows significant sequence homology withmelittin in the regions around amino acids 131 and 132, amino acids191-193, and amino acids 260 through 280. The amino acid sequence ofmelittin and the predicted amino acid sequence of PLAP were comparedusing the Wisconsin database software (GCG). A comparison of the aminoacid sequence of melittin and amino acids 260-280 of PLAP is shown inTable 1. At the present time fragments of PLAP selected to contain atleast a portion of the amino acids in one of these regions is preferred,more preferably the fragment contains at least half to all of the aminoacids in one of these regions. Fragments may be selected by using one ofthe aforementioned homologous regions as a starting point and proceedingeither towards the animo terminus or the carboxy terminus of PLAP forthe desired number of amino acids, and constructing a fragment orpeptide having the corresponding amino acid sequence. Examples ofpeptides of varying length may be found in FIG. 1. These peptides wereselected from the regions of PLAP having homology with mellitin.Applicants have found that fragments (or peptides) selected from thisarea have PLA₂ stimulating activity as shown in Table 2. Also at thepresent time, preferred fragments are those having greatest amounts ofPLA₂ stimulating activity. Fragments of PLAP selected outside theaforementioned areas that have PLA₂ stimulating activity are also withinthe scope of the invention. Peptides 26 amino acids in length having thesequence of the amino or carboxy terminus of PLAP have been tested, butwere not found to have PLA₂ stimulating activity.

Fragments of PLAP may be prepared by any method for preparing peptidesincluding chemical synthesis, recombinant DNA techniques, anddegradation of PLAP. Chemical synthesis of the fragments or peptides ispresently preferred for convenience of preparation. The fragments orpeptides may be synthesized by any convenient method for synthesizingpeptides or proteins.

                  TABLE 1                                                         ______________________________________                                        Melittin                                                                             GlyIleGlyAlaValLeuLysValLeuThrThrGlyLeuProAla                          PLAP   GluSerProLeuIleAlaLysValLeuThrThrGlu - ProPro                          Melittin                                                                             LeuIleSerTrpIleLysArgLysArgGlnGln                                      PLAP   IleIleThrProValArgArg                                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Peptide  Phospholipase A.sub.2 activating activity                            ______________________________________                                        A        ++                                                                   D        -                                                                    F        ±±                                                             G        ±                                                                 1        +                                                                    2        +                                                                    3        +                                                                    4        +                                                                    ______________________________________                                         ++ indicates PLA.sub.2 stmulating activity approximately 5 times greater      than controls; + indicates 2-5 fold stimulation of PLA.sub.2 activity whe     compared to controls; ± and ±± indicate inconclusive results for     PLA.sub.2 stimulating activity; - indicates no PLA.sub.2 stmulating           activity.                                                                

A nucleic acid sequence coding for PLAP isolated from the murine BC3H1cell line as described herein is shown in FIG. 2. Fragments,subsequences, additions and deletions of bases from the sequence thatcode for PLAP fragments, portions, or derivatives capable of serving asantigen for production of antibodies specific for PLAP, whetherconjugated to a carrier protein or not, or that code for a portion ofPLAP having PLA₂ stimulating activity are also within the scope of theinvention.

For diagnosing and following the course of rheumatoid arthritis,synovial fluid is removed from at least one affected joint of a patientsuspected of having rheumatoid arthritis. The synovial fluid is thentested to detect the presence of elevated levels of PLAP which wouldsignify rheumatoid arthritis. The synovial fluid is tested usingimmunoassays described herein in another embodiment of the invention.The immunoassays of the invention may be used in combination with othersymptoms of the patient to diagnose the disease. Generally theimmunoassays will be performed by physicians or other trained medicalpersonnel after a physician has examined the person and suspects thatthe person has rheumatoid arthritis based on the physical symproms ofthe person. The symptoms of rheumatoid arthritis are well-known tophysicians. Once the physician suspects the person has rheumatoidarthritis, the immunoassays of the invention may be performed to rule inor rule out the presence of the disease in the person. The physicalmanifestations of rheumatoid arthritis are sometimes difficult todifferentiate from other diseases, such as osteoarthritis, pseudogout,and seronegative spondyloarthropathy. The immunoassays of the inventionwill allow the physician to correctly diagnose the disease, andprescribe appropriate treatment. Thus the immunoassays of the inventionare also useful for ruling out the presence of rheumatoid arthritis inpersons suspected of having the disease.

Since normal human, and other mammalian synovial fluid and tissuescontain PLAP, it may be necessary, in order to determine elevated levelsof PLAP to obtain baseline or background levels of PLAP, and tocalibrate a particular embodiment of the immunoassays of the inventionto take into account the presence of PLAP in healthy persons not havingrheumatoid arthritis. For example, the concentration of PLAP in a sampleof persons not having rheumatoid arthritis would be determined and andused to compare with the amount of PLAP found in a sample from a personsuspected of having rheumatoid arthritis. If the amount of PLAP in thesample from the person suspected of having rheumatoid arthritis isgreater than the baseline or background levels found in the healthypersons, the increased amount would indicate the presence of rheumatoidarthritis in the person. Similarly, the value for elevated levels ofPLAP may be calibrated by testing persons known to have rheumatoidarthritis to determine elevated levels of PLAP for a particularembodiment of the immunoassays of the invention. For example, commonlyused detectable labels such as enzymes use different colorimetricsubstrates that are detected by spectroscopy at different wavelengths.Results are typically read as optical density at a certain wavelength.Thus a baseline amount of PLAP would be established as a the opticaldensity present when the synovial fluid of healthy persosn is tested.Elevated levels would be established in a similar manner. Once baselinelevels and elevated levels are established, the significance of apatient's result will be routine to establish. The level of PLAP in asample from a person suspected of having rheumatoid arthritis isdetermined using an immunoassay of the invention, and the result iscompared with the baseline, and possibly elevated levels for theimmunoassay. If the sample contains elevated levels of PLAP, the personvery likely has rheumatoid arthritis. If the level falls between theelevated level and the baseline level, a diagnosis of rheumatoidarthritis is not as likely. In addition to calibration for a particulartype of immunoassay, it may also be necessary to establish baseline andelevated levels of PLAP for a particular type of tissue. All suchmanipulations are within the scope of one skilled in the art.

Synovial fluids from persons having rheumatoid arthritis consistentlycontain very high levels of PLA₂ activity when compared to synovialfluid from normal patient joints or even the serum from rheumatoidarthritic patients. In contrast synovial fluid from inflamedosteoarthritic joints shows only slightly elevated levels ofphospholipase activity.

The immunoassays of the invention may also be used to follow the courseof rheumatoid arthritis. Since, rheumatoid arthritis is a long termchronic disease for which there is at the present time no cure, it wouldbe useful to have assays to determine the effectiveness of therapeuticagents used in treating the disease, and possibly in determiningflare-ups or remissions of the symptoms. The immunoassays of theinvention may accordingly be performed at predetermined intervals orwhen desired by the physician or patient to determine the level of PLAPin the synovial fluid of the person having the disease. The level ofPLAP in the synovial fluid should give an indication of the state of thedisease.

Synovial fluid or other body fluid or tissue may contain components thatcould interfere with the performance of the immunoassays of theinvention and which may need to be removed prior to use. Synovial fluidis preferably centrifuged prior to use in the immunoassay, and theclarified fluid is used. Similarly, when body tissues are used, thetissues are preferably ground into small pieces, such as in a blender,then centrifuged to separate the pieces of tissue, and supernatant usedin the immunoassays of the invention.

The immunoassays of the invention may be performed using a conventionalimmunoassay format such as antibody capture, antigen capture, ortwo-antibody sandwich assays. Immunoassay formats may be found instandard texts such as Harlow and Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988. In apreferred immunoassay, synovial fluid suspected of containing PLAP isdeposited in a multi-well microtiter plate. Antibodies specific for PLAPare then contacted with the components of the synovial fluid, includingPLAP if present. Bound antibody is then contacted with second adetectably labeled antibody capable of binding with the antibodyspecific for PLAP, and the detectable label is detected, therebysignifying the presence of PLAP in the synovial fluid. Alternatively,anti-PLAP antibodies could be deposited in wells of a microtiter plateand synovial fluid contacted with the deposited antibodies where PLAP inthe synovial fluid would bind to the deposited antibodies. Bound PLAPcould then be detected by binding a detectably labeled anti-PLAPantibody to the bound PLAP, and subsequently detecting the detectablelabel. Other immunoassays may be readily designed to detect PLAP byreference to standard texts in the field referred to above. As usedherein, the term antibody is intended to mean antibodies, and anyfragments or portions of antibodies that are capable of binding to PLAP.

Immunoassays may also be used to detect the presence of PLAP in tissue,such as joint tissue, in situ. Tissue specimens may obtained andprepared according to conventional methods for preparing tissues.Antibodies specific for PLAP are then contacted with the prepared tissuewhere the antibodies will bind to PLAP in the tissue. Bound antibodiesare then detected.

Suitable detectable labels for use in the immunoassays of the inventioninclude enzymes such as horseradish peroxidase and alkaline phosphatase,radiolabels such as ¹²³ I and ³² P, and biotin systems, latex particles,electron dense materials such as ferritin, light scattering materialssuch as gold. Suitable detectable labels for use in the immunoassays ofthe invention may be found in Harlow and Lane, Antibodies, A LaboratoryManual, supra and other standard texts in the field. Suitable methodsfor detecting the detectable label include scintillation counting,autoradiography, fluorescence measurement, calorimetric measurement, orlight emission measurement.

Thus, the labeling may comprise a radiolabel (e.g. ¹⁴ C, ³² P, ³ H, andthe like), an enzyme (e.g., peroxidase, alkaline or acid phosphatase,and the like), a bacterial label, a fluorescent label, an antibody(which may be used in a double antibody system), an antigen (to be usedwith a labeled antibody), a small molecule such as biotin (to be usedwith an avidin, streptavidin, or antibiotin system), a latex particle(to be used in a buoyancy or latex agglutination system), an electrondense compound such as ferritin (to be used with electron microscopy),or a light scattering particle such as colloidal gold, or anycombinations or permutations of the foregoing.

For ease in performance of the immunoassays of the invention, theimmunoassays may be provided in a kit form. The kits of the inventioncomprise an solid surface, such as a multi-well titer plate,nitrocellulose strip, tissue culture plates or other apparatus forcontaining or binding PLAP or anti-PLAP antibodies, and one or morereagents for detecting PLAP. The kits may also comprise one or morereagents for standards or controls. For example, in a preferredembodiment, the kits may comprise a multi-well plate for depositing thesynovial fluid sample, a reagent comprising antibody specific for PLAP,a reagent comprising enzyme-labeled antibody bindable with the antibodyspecific for PLAP, and a reagent comprising substrate for the enzyme.The kits of the invention will vary according to the format of theimmunoassay used to detect PLAP. If the immunoassay is to be performedin a liquid, the kits may comprise one or more containers for performingthe assay, and one or more reagents for detecting PLAP. The reagents ofthe invention comprise antibodies specific for PLAP or a PLA₂stimulating portion thereof and a carrier or diluent. Suitable carriersor diluents include water, and saline, or other buffer. The reagents mayalso comprise preservatives or stabilizers. The reagents of theinvention may be supplied without the carrier or diluent, such as wouldbe the case if the reagent were freeze-dried. A suitable amount of thecarrier or diluent would then be added by prior to using the reagent.

Polyclonal and monoclonal antibodies specific for PLAP or fragments ofPLAP may be prepared by standard techniques in the field such as may befound in Harlow and Lane, Antibodies, A Laboratory Manual, supra andother standard texts in the field. To prepare polyclonal antibodies,purified PLAP, PLAP produced by recombinant DNA techniques (rPLAP) orfragments of purified PLAP or rPLAP are injected into an animal such asa rabbit, or mouse. Antibodies produced by the animal in response toPLAP or fragment are then purified from the serum of the animal. Sincesome of the fragments of purified PLAP or rPLAP may not be large enoughto produce a strong immune response in the animal, they may beconjugated to a carrier protein such as keyhole limpet hemocyanin orbovine serum albumin prior to injection into the animal. Monoclonalantibodies may be produced using the method of Kohler and Milstein,Nature 256: 495-497 (1975), or as described in standard texts in thefield such as Harlow and Lane, Antibodies, A Laboratory Manual, supra.

Experimental Materials and Methods

Radioactive materials were purchased from New England Nuclear (Boston,Mass.). Tissue culture media and serum were from Hazelton (Denver, Pa.).Tissue culture plates, antibiotics, protease inhibitors and deoxycholatewere from Sigma (St. Louis, Mo.). Thin layer chromatography plates werefrom Analtech (Newark, Del.). Authentic phospholipid standards were fromAvanti Lipids (Birmingham, Ala.). Protein determinations were performedaccording to the method of Bradford according to Bio-rad instructions(Richmond, Calif.). Peroxidase-conjugated goat anti-rabbit lgGantibodies were from Cappel (Malvern, Pa.). Anti-actin antibodies werefrom Amersham (Arlington Height, Ill.). Scintillation fluid and silicabased affinity supports were from Beckman Instruments (Fullerton,Calif.). Synthetic melittin was from Penninsula Laboratories (Belmont,Calif.). Iodo-beads were from Pierce (Rockford, Ill.).

Patients

All patients with rheumatoid arthritis presented definite or classicalsymptoms as defined by the American Rheumatism Association (Ropes (1968)Bull. Rheum. Dis. 9: 175-176). Disease severity was assessed by thestandard roentgenographic and clinical criteria of Steinbrocker et al.(1949) JAMA 140: 659-662. Progression of rheumatoid arthritis wasdefined as stage 1 or or early on the basis of no destructive changes onroentgenographic examination. Stage 2 or moderate disease was defined asroentgenographic evidence of osteoporosis without joint deformities.Stage 3 or severe disease was defined on the basis of roentgenograpgicevidence of cartilage and bone destruction with joint deformity. Stage 4or terminal disease was defined on the basis of fibrous or bonyankylosis. Functional capacity was defined as class 1 with completefunctional capacity, class 2 as functional capacity adequate to performnormal activities despite discomfort or limited mobility and class 3 asable to perform few or no activities associated with occupational ornormal life routines. Class 4 was defined as complete immobilizationwith the patient bedridden or wheelchair bound. Control patients withosteoarthritis (non-inflammatory arthritis) and other arthropathies alsomet American Rheumatism Association criteria (Huth (1986) Ann. Intern.Med. 102: 553. Normal volunteers were also used and were free ofprescription medication or other drugs.

Table 3 shows clinical characteristics of rheumatoid arthritis patients.

                  TABLE 3                                                         ______________________________________                                        CLINICAL CHARACTERISTICS OF RHEUMATOID                                        ARTHRITIS PATIENTS*                                                                               Mean (±SD)                                             ______________________________________                                        Age (years)           53.5 ± 14.1                                          Sex                   28 Male, 28 Female                                      Disease Duration (months)                                                                           110.6 ± 113.7                                        Synovial Fluid WBC (cells/mm3)                                                                      24,620 ± 6633                                        NSAID                 53 Yes                                                  DMARD                 37 Yes                                                  Prednisone            18 Yes                                                  Westergren Sedimentation Rate                                                                       54.5 ± 39                                            Rheumatoid Factor (titer)                                                                           1:1405                                                  Number of A.C.R. Criteria                                                                           8.1 ± 1.1                                            Stage of Progression  2.2 ± 0.8                                            Functional Class      2.5 ± 0.6                                            ______________________________________                                         *Snyovial fluid samples from 56 patients were examined. The abbreviation      are NSAID (nonsteriodal antiinflammatory drug), DMARD (disease modifying      antirheumatic drug) and ACR (Americah College of Rheumatology). All           patients on prednisone were on 10 mg/day or less. Fourteen pateints were      rheumatoid factor negative. The mediam rheumatoid factor titer in patents     in whom it was present ws 1:640, although the mean of all positive            patenitsn were 1:1405. The A.C.R. Criteria and Stage of Progression and       Functional Class are as noted above.                                     

Synovial Fluid

Synovial fluid was obtained from Arthritis Center Clinical Laboratories(Veterans Administration Medical Center, Philadelphia, Pa. andRheumatology Division, Bowman Grey School of Medicine, Winston-SalemN.C. after white cell count, gram stain, and examination using polarizedlight microscopy. Fluids were centrifuged to remove any cells and storedat -70° C.

Definition of PLAP Activity

Fractions collected during affinity or other chromatography steps wereassayed as described in Clark M. A. et al. (1987) J. Biol. Chem. 262:4402-4406, the disclosures of which are hereby incorporated by referenceas if fully set forth herein. Twenty microliter aliquots of eachfraction were assayed in triplicate for endogenous phospholipase A₂(PLA₂ ) and phospholipase C (PLC) activity in human U937 cell freesonicates and for the ability to stimulate PLA₂ activity above controls.PLAP stimulatory activity was defined as the difference between theactivity observed with cell free sonicates and endogenous activityobserved for each individual fraction. One unit of stimulatory activitywas defined as the amount of PLAP required to produce a 2-fold increasein measured PLA₂ activity per 1 mg of U937 cell free sonicate protein.PLAP purified from synovial fluid was stable for several weeks at -70°C. and could be frozen and thawed 3 times without loss of activity.

Phospholipase Assays

Phospholipase A₂ (PLA₂) and phospholipase C (PLC) activities werequantified radiometrically as described in Clark M. A. et al. (1981) J.Biol. Chem. 262: 10713-10718; Bomalaski et al. (1985) J. Leukocyte Biol.38: 649-654; and clark et al. (1986) J. Biol. chem. 261: 10713-10718,the disclosures of all of which are hereby incorporated by reference asif fully set forth herein, except that reactions were buffered with 200mM Tris pH 9.0. For PLA₂ and PLC formation of lysophospholipid anddiglyceride generation was measured respectively.

ELISA Assay

Twenty microliters per well of synovial fluid was deposited in wells ofa microtiter plate with each well then being filled to capacity withphosphate buffered saline (PBS) containing 20% fetal calf serum. Theplates were then incubated at 4° C. overnight. Rabbit anti-melittin oranti-PLAP (recombinant or purified protein or fragments there-of)antibodies were diluted 1-1000 in PBS and 50 ul added per well. Forcontrol experiments anti-actin antibodies diluted 1-1000 weresubstituted for the anti-mellittin or anti-PLAP antibodies. Neitheranti-actin nor pre-immune antisera showed immunopositive reactions withsynovial fluid from normal volunteers or arthritic patients. Afterovernight incubation with antibodies, plates were further incubated atroom temperature for 1 hour and then rinsed 3 times (5 minutes each)with PBS. 50 μl of peroxidase conjugated goat anti-rabbit antibody(diluted 1-5000 in PBS) was then added to each well followed by a 1 hourincubation at room temperature and 3 washes (5 minutes each).O-phenyleuediamine (a peroxidase substrate) was added (100 μl of a 1mg/ml in 0.1M citrate buffer, pH 4.5, 0.12% H₂ O₂ solution) and allowedto react for 10 minutes. The reaction was stopped by the addition of 25μl of NaF (0.1M) to each well. Absorbance for each well was determinedusing a Titertek Multiscan model MC (Helsinki, Finland) ELISA platereader at 490 nm. Anti-melittin or PLAP antibodies did not cross reactwith other molecules, including complement cascade components.

In order to quantitate the presence of PLAP as a function of thepresence and stage of rheumatiod arthritic disease ELISA assays wereperformed on a broad spectrum of synovial fluid samples drawn fromrheumatoid arthritic patients, normal healthy volunteers, and patientswith other form of arthritis or joint disease. This data is summarizedin Table 4. As shown specimens from patients with rheumatoid arthritisshowed an average 4.3-fold increase in PLAP levels over healthy synovialfluid or fluid from patients with osteoarthritis. Within the detectionlimits of the assay 87% of rheumatoid arthritic patient synovial fluidshowed elevated PLAP levels. Elevated levels of PLAP were detected inrheumatoid arthritic patients and increased levels of the activatingprotein were associated with severity of the disease (p<0.05).

                  TABLE 4                                                         ______________________________________                                        ELISA Assay for PLAP*                                                         Arthritis                                                                     Value         Number    ELISA Value  p                                        ______________________________________                                        Rheumatoid    32        1.88 ± 1.19                                                                             --                                       --                                                                            Osteoarthritis                                                                              12        0.44 ± 0.62                                                                             0.05                                     Pseudogout    5         0.20 ± 0.44                                                                             0.05                                     Seronegative  5         0.28 ± 0.68                                                                             0.05                                     Spondyloarthropathy                                                           ______________________________________                                         *Expressed as optical density (o.d.)                                     

For experiments using BC3H1 smooth muscle cells, cells were grown in 96well plates to 75% confluence over 2-3 days and treated in quadruplicatewith leukotriene D4 (1 μM) for varying time periods. Reactions werestopped by the addition of formalin to a final concentration of 3% V/V.Cells were permeabilized with 0.05% Tween 20 and reacted withanti-melittin antibodies (diluted 1-100) for 1 hour. Quantification ofbound antibody was performed using a peroxidase conjugated secondantibody (Malloy Labs) according to manufacturers' instructions.

Localization of PLAP in Synovial and Rheumatoid Nodule Tissue

Synovial tissue from 6 patients with definite or classical rheumatoidarthritis and 6 patients with osteoarthritis who had undergone totaljoint replacement and subcutaneous nodules from 2 rheumatoid patientswith classical disease were processed by routine histologic methods forpathological examination. Bone specimens were prepared calcified anddecalcified. Immunochemical localization of PLAP was performed usinganti-melittin or anti-PLAP antibodies.

Isolation of PLAP from Human Synovial Fluid

PLAP isolation from human synovial fluid was performed essentially asdescribed in Clark M. A. et al. (1987) J. Biol. Chem. 262: 4402-4406,the disclosures of which are hereby incorporated by reference as iffully set forth herein. Affinity purified anti-melittin antibodies inPBS were immobilized on activated affinity silica supports and thecolumns equilibrated with PBS containing the protease inhibitorsphenymethylsulfonyl fluoride (10 uM) bacitracin (100 μg/ml), benzamidine(1 mM), soybean trypsin inhibitor (5 g/ml) and Tween 20 (0.05%). Priorto chromatography synovial fluid was centrifuged at 13,000×g for 10minutes. The resulting supernatant was diluted 1:1 with PBS and appliedto the silica column at a flow rate of 0.1 ml/minute at roomtemperature. The column was washed for 5 minutes (2 ml/minute) with PBSand eluted with 50 mM sodium acetate pH 3.1 (0.5 ml/minute). Fractionscontaining PLAP activity were pooled and further purified by HPLC sizeexclusion chromatography using a TSK3000 sw HPLC column equilibratedwith PBS containing 0.05% Tween 20 at a flow rate of 0.5 ml/min. SDSpolyacrylamide gel electrophoresis using 15% T gels was performed toassess protein purity. Samples for electrophoresis were iodinated using¹²⁵ I and Iodobeads (Pierce) according to the manufacturers'instructions.

No lipopolysaccharide contaminants were observed in purified PLAPfractions used for in vivo testing as measured by the limulus amibocyteassay (detection limit 0.1 5 ng/ml).

Purification of PLAP from human synovial fluid by affinitychromatography using silica immobilized anti-melittin antibodies isillustrated in FIG. 3. Purification of PLAP to homogeneity by sizeexclusion HPLC using a silica based size exclusion column is illustratedin FIG. 4. Fractions were assayed for PLAP activity as described herein.An aliquot of pooled and concentrated fractions were iodinated using ¹²⁵I and subjected to SDS polyacrylamide gel electrophoresis. A single bandat 28,000 molecular mass was observed on autoradiography of theelectrophoresis gel. The estimated molecular size of the protein fromthe size exclusion column (43,000 molecular mass) may reflect anartifact due to the presence of detergents or an oligomeric structurefor PLAP. Similar results were obtained using anti-rPLAP antibodiesalthough silica is the preferred support for immobilization due to lowersample dilution.

Phospholipase Specificity Of PLAP Isolated From Human Synovial Fluid

The phospholipase specificity of PLAP was examined using the majorphospholipids listed in Table 5. Rheumatoid synovial fluid PLAP wasfound to be specific for phosphatidylcholine preferring PLA₂ and did notaffect phospatidylethanolamine or phosphatidylinositol activities. PLCactivity was not affected by PLAP. These results are qualitativelysimilar to PLAP isolated from other mammalian sources (Clark et. al.(1987) J. Biol. Chem. 262: 4402-4406 and Clark et al. (1988) Biochem. J.250: 125-132. rPLAP isolated as a fusion protein was devoid ofphospholipase stimulatory activity.

                  TABLE 5                                                         ______________________________________                                        Phospholipid Substrate Specificity of PLAP*                                                 Sonicate                                                                             PLAP    Sonicate plus                                                  alone  alone   PLAP (1 unit)                                    ______________________________________                                        Phospholipase A.sub.2                                                         Phosphatidylcholine                                                                           7.9 ± 0.2                                                                           BG      16.1 ± 1.9*                               Phosphatidylethanolomine                                                                      2.5 ± 0.5                                                                           BG      2.0 ± 0.3                                 Phosphatidylinositol                                                                          <0.1     BG      <0.1                                         Phospholipase C                                                               Phosphatidylcholine                                                                           1.9 ± 0.3                                                                           BG      2.1 ± 0.2                                 Phosphatidylinositol                                                                          0.6 ± 0.2                                                                           BG      0.6 ± 0.1                                 ______________________________________                                         *Expressed as pmol of product produced/mg cell protein using U937             monocytes as a source of PLA.sub.2. One unit of PLAP was defined as that      amount of PLAP required to produce a 2fold increae in the observed            phospholipase A.sub.2 activity found per mg protein of U937 cells, and wa     diluted to a final volume of one unit of PLAP per 10 ul of buffer. BG --      background. Boiled PLAP had no phospholipase A.sub.2 stimulatory activity     **p < 0.05 compared to sonicate alone by Student's ttest.                

Cell Culture

Human monocyte U937 cells were a gift from Dr. Giorgio Trinchieri of theWistar Institute (Philadelphia, Pa.) or were purchased from the AmericanType Tissue Culture Collection (Bethesda, Md.) accession number CRL1593, and were maintained in RPMl 1640 supplemented with 10% heatinactivated fetal calf serum. Cells were used during the log growthphase. BC3H1 murine smooth muscle cells and CPAE bovine endothelial celllines were also obtained from the American Type Tissue CultureCollection accession numbers CRL 1443 and CRL 209, respectively. Cellswere grown in Dulbecco's Modified Minimal Essential Media containing 20%fetal calf serum in 95% air, 5%CO₂.

Injection of PLAP and PLAP Derived Synthetic Peptides into Rabbit Joints

Male New Zealand white rabbits weighing 3-4 kg (3 per group) wereanesthetized and prepared for surgery according to standard protocols.Rabbits were injected in the knee joint with 1 ml of sterile pyrogenfree purified synovial fluid PLAP (containing 3000-100000 units ofsaline) and in the contralateral knee with an equal volume of pyrogenfree saline or saline containing 0.15 ng/ml lipopolysaccharide or 10μg/ml actin. Additional controls were pooled size exclusion HPLCfractions devoid of PLAP activity concentrated and processed as for PLAPor boiled denatured PLAP preparations. Injections were made through thelateral joint capsule so as to avoid the infrapatellar fat pad. Twentyfour hours post-injection the rabbits were necropsied and both kneeslavaged with 2 ml of pyrogen free saline prior to opening. The lavagefluid was cultured for bacteria and the total and differential cellcount determined. An aliquot of the lavage fluid was centrifuged and thesupernatant frozen and stored at -70° C. until radio-immunoassay, usingradioimmune assay kits (New England Nuclear, Boston, Mass.) according toinstructions, for prostaglandin E₂ and leukotriene C₄. The synoviallined infrapatellar fat pad portion from each joint was also excised andfixed in formalin and embedded in paraffin for routine histology.Bacterial cultures of synovial fluids were negative.

PLAP stimulation of an inflammatory arthritogenic response was examinedby injection of purified PLAP into rabbit knee joints. Saline, boiled(denatured) PLAP as well as the protein controls described above wereinjected into the contra lateral joint. The dose-response relationshipof PLAP in mediating an inflammatory arthropathy with cellularinfiltration and joint destruction is summarized in Table 6. After 24hours post-injection of (3000-50000 PLAP units) the total cell count ofsynovial lavage fluids was dramatically increased. Although the majorityof inflammatory cells present were polymorphonuclear leukocytes (95%PMN), there was an absolute increase in mononuclear inflammatory cellsas well. After 24 hours post-injection with 3000 PLAP units fewer PMNand more monocytes were observed.

Histologically, with up to 50000 PLAP units there was a dose relatedinflammatory cell infiltration of the infrapatellar fat pad primarilylocated immediately beneath and within the synovial lining. Synovialproliferation was also observed. After injection of 100,000 PLAP units,the inflammatory cell infiltrate was markedly reduced. This reductionwas associated with hemorrage, necrosis and loss of synovial liningcells and further accompanied by multifocal necrosis and hemorrage ofthe tunica media arterioles. These data demonstrate the ability of PLAPto mediate synovial fluid leukocytosis and inflammatory synovitis withmild inflammation to gross tissue damage depending on dose.

When PGE2 levels were measured in lavage fluid post-injection of 50,000PLAP units a slight increase was observed over controls (0.1 ng/ml). NoLTC4 was detected. In control experiments, no tissue damage orinflammatory cell infiltration was observed.

                  TABLE 6                                                         ______________________________________                                        Induction of Synovial Fluid Leukocytosis in Rabbits*                          Compound Injected      WBC (×10.sup.5)/ml                               ______________________________________                                        Actin (10 ug/ml)        1.1 ± 0.2                                          LPS (0.15 ng/ml)        9.1 ± 0.1                                          HPLC Eluent            12.3 ± 2.1                                          Boiled PLAP (60,000 units/ml)                                                                        10.8 ± 0.8                                          PLAP (3,000 units/ml)  38.6 ± 15.4**                                       PLAP (6,000 units/ml)  51.2 ± 8.1**                                        PLAP (25,000 units/ml) 68.3 ± 16.4**                                       PLAP (50,000 units/ml) 73.5 ± 13.5**                                       PLAP (100,000 units/ml)                                                                               6.2 ± 2.9                                          ______________________________________                                         *PLAP isolated from the HPLC gel filtration column was injected into          rabbit knee joints. Control joints were injected with actin, LPS or HPLC      gel filtration eluent. The amount of LPS injected was the amount              detectable by this assay. Eluent from HPLC fractions without PLAP acitivy     was concentrated 16fold prior to injection. After twenty four hours, the      rabbits were necropsied and the knee synovial cavity was lavaged with 2 m     of phosphate buffered saline.                                                 **p < 0.05 by Student's ttest compared to HPLC eluent or boiled PLAP.    

Uptake and Release of (3H) Arachidonic Acid

Normal human peripheral blood monocytes in 24 well tissue culture plateswere incubated with labelled arachidonic acid for 24 hours as describedin Bomalaski J. (1986) Clin. Immunol. Immuopath. 39: 198-212, thedisclosures of which are hereby incorporated by reference as if fullyset forth herein. The mononuclear leukocytes were then separated usingFicoll-Hypaque according to conventional methods, washed, resuspended inRPMl-1640 media containing 10% heat inactivated fetal calf serum(complete media) and allowed to adhere for 1 hour. Nonadherent cellswere removed by a wash with PBS and overlayed with labelled arachidonicacid for 24 hours. Cells were washed twice with PBS and reincubated withcomplete media or complete media containing 10 units/ml PLAP. Atappropriate time points aliquots of cell supernatant were taken andcounted by scintillation spectrometry. Cell viability was determined bytrypan blue dye exclusion. Lactate dehydrogenase release assays showedidentical results for control and treated cells.

Induction of Eicosanoid Generation in Peripheral Blood Cells

Human peripheral blood monocytes and reticulocytes were prepared bystandard methods. Cells were incubated with PLAP for various times,centrifuged and the supernatants recovered for assay. Usingradioimmnoassay kits (New England Nuclear, Boston, Mass.) leukotriene B₄and prostaglandin E₂ release were measured.

To demonstrate PLAP induction of eicosinoid release, normal humanperipheral blood monocytes preloaded with labelled arachidonic acid werestimulated with PLAP. A signifigant release of arachidonic acidmetabolites was observed as summarized in FIG. 5. Radioimmunoassay forspecific metabolites of unlabelled cells showed prostaglandin E₂ as themajor metabolite released as shown in Table 7. Similarly PLAP was shownto stimulate release of leukotriene B₄ and prostaglandin E₂ (Table 7).

                  TABLE 7                                                         ______________________________________                                        PLAP and PHOSPHOLIPASE                                                        A.sub.2 STIMULATE EICOSANOID RELEASE*                                                   Human Monocyte                                                                            Human Neutrophil                                                  Prostaglandin E.sub.2                                                                     Leukotriene B.sub.4                                               (pg/2.5 × 10.sup.6 cells)                                                           (pg/2.5 × 10.sup.6 cells)                                   Time (min)  Time (min)                                              Treatment   5        20       5       20                                      ______________________________________                                        Buffer      0        2         5      21                                      PLAP (1 unit)                                                                             2        7        13      28                                      Metlittin (10 ug)                                                                         28       71       28      41                                      PLA.sub.2 - Porcine                                                                       0        7        167     >300                                    (14 pmol)                                                                     PLA.sub.2 - Bovine                                                                        7        30       234     >300                                    (14 pmol)                                                                     ______________________________________                                         *Human peripheral blood monocytes and neutrophils were prepared as            described. Cells were placed in 1 ml microfuge tubes and incubated at         37° C. for five or twenty minutes with the indicated compounds. Th     tubes were then centrifuged, and the supernatants taken for eicosanoid        determination by radioimmunoassay. Each sample was prepared in duplicate,     and is expressed as the mean value; standard deviation was within 10%.        Data from a representative experiment of which three were performed.     

Localization of PLAP in Rheumatoid Synovial and Subcutaneous NoduleTissue

To determine the cellular source of PLAP found in human joints,metacarpophalangeal and knee joint specimens were obtained from patientswith rhematoid arthritis or osteoarthritis and immunohistochemicalstaining with anti-PLAP antibodies performed. All 6 rheumatoid arthriticpatients tested displayed intense staining. No staining was observedwith the specimens obtained from osteoarthritic patients.

Microscopic examination revealed heavy staining of synovial monocytes,macrophages, and multinucleated giant cells with some additionalstaining of vascular smooth muscle and endothelial cells. Similarresults were observed in rheumatoid nodules. Osteoarthritic synoviumrevealed no PLAP staining even under microscopic examination. Nostaining was observed in either speciment type with pre-immune antisera.

Statistical Data Analysis

Data was analyzed using the students t-test or where appropriate theSpearman linear rank correlation coefficient or Wilcoxon correlationusing as SSPS Inc. Data Programming Model (Chicago, Ill.). The number ofsamples required to avoid a Type 2 error was determined as described inYoung (1983) Annal. Intern. Med. 99: 248-251. For the animal data arandomized complete block analysis of variance LSD test was used tocompare differences between the treatment means. An alpha of 0.05 wasused as a criteria for signifigance (SAS Manual).

Molecular Cloning of PLAP

Unless otherwise stated, purification and cloning was performedaccording to standard techniques such as those found in Sambrook, etal., Molecular Cloning, A Laboratory Manual, second edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

BC3H1 cells were treated with leukotriene LTD₄ (1 μM) for 2 minutesprior to extraction of total cellular mRNA with quanidine isothiocyanateto increase synthesis of mRNA coding for PLAP. PolyA containing RNA wasselected by affinity chromatography on oligo(dT) cellulose.

A cDNA library was then prepared utilizing the polyA selected RNA. Thefirst strand of cDNA was synthesized by reverse transcriptase using anoligo dT primer. The second strand of cDNA was synthesized using DNApolymerase I and E. coli DNA ligase according to the method of Gublerand Hoffman (1983) Gene 25: 263-279. EcoRl linkers (Vector Labs,Burlingame, Calif.) were added and the inserts ligated to λgt11 (VectorLabs, Burlingame, Calif.) according to the manufacturers' protocol. Theresulting library was screened to identify clones containing DNA codingfor PLAP according to the method of Young and Davis (1983) Proc. Natl.Acad. Sci. USA 80: 1194-1199, and Snyder, M. et al. Method Enzymol. 154:107-128 (1984). Anti-melittin antibodies were prepared according to themethod of Clark et al. (1988) Biochem. J. 250: 125-132, and Clark et al.(1987) J. Biol. Chem. 262: 4402-4406, the disclosures of both of whichare hereby incorporated by reference as if fully set forth herein.

The positive cDNA clone identified by screening with anti-mellitinantibodies was subcloned into M13 and sequenced using the dideoxy methoddescribed in Sanger et al., (1980) J. Mol. Biol. 143: 161-169, andSnyder, M. et al., supra. Enzymes and other necessary reagents wereobtained from U.S. Biochemical Corporation, Cleveland, Ohio. Thesequence of this clone is shown in FIG. 2.

Expression of rPLAP in BC3H1 Cells

The metallothionein promoter described by Brinster et al. Nature 296:39-42 was chemically synthesized and ligated into the plasmid pUC19(Southern and Berg (1982) J. Mol. Appl. 1: 327-341) containing aneomycin resistance gene. The cDNA coding for PLAP was excised from thelambda gt 11 clone using EcorRl and ligated into pUC19 downstream fromthe metallothionein promoter.

The PLAP pUC19 expression vector was transfected into BC3H1 cells byelectroporation using 100 μg of DNA/10⁶ cells and a BioRadelectroporation device using the setting of 250 volts and 25 μFd. Cellswere grown in Dulbecco's Modified Minimal Essential Medium (GIBCO, GrandIsland, N.Y.). Stable transformed cells were then selected by growth inmedium supplemented with neomycin (0.3 mM) (G418, GIBCO, Grand Island,N.Y.). Transformed cells were incubated overnight in [³ H]-arachidonicacid (10 μCi/ml). The next day the cells were rinsed three times insaline before being treated with copper. Aliquots of the supernatant (1ml) were collected at predetermined times and the amount ofradioactivity released was determined by liquid scintillationspectroscopy. Clones containing the PLAP cDNA insert in the 5' to 3'orientation increased the rate of release of radioactive arachidonicacid acid metabolite following treatment with copper, whereas cellswhich contained the PLAP cDNA clone in the 3' to 5' orientation appearedto suppress the release of [³ H]-arachidonic acid and its metabolites.

Antibody Production Using Recombinant PLAP (rPLAP)

Antibodies were generated to recombinant PLAP (rPLAP) prepared as aβ-gal-PLAP fusion protein. The β-gal fusion protein was preparedaccording to the method disclosed in Snyder, M. et al. (1984) Methods inEnzymology 154: 107-128, the disclosures of which are herebyincorporated by reference as if fully set forth herein. Briefly, E. coli1089 cells were infected with the lambda clone containing the PLAP cDNAand producing lysogens. Next the cells were grown at 30° C. to a OD₆₀₀of 0.4 and the synthesis of the β-gal PLAP fusion protein induced by ashift to 37° C. in the presence of isopropyl-B-D-thiogalactoside (IPTG,100 μg/ml) for 1 hour. The cells were then harvested, placed in SDSsample buffer [Laemmli, U. K. (1970) Nature 227: 680-685] and frozen.The fusion protein isolated by preparative SDS polyacrylamideelectrophoresis using a 6% polyacrylamide gel. The band containing thefusion protein was identified by its increased synthesis in response toIPTG treatment and by Western blot analysis using anti-melittinantibodies. The band, which was well-resolved from other proteins, wasexcised and crushed, mixed with adjuvant and injected subcutaneouslyinto female New Zealand White rabbits (100 μg/injection) according tostandard protocols. Control antisera to β-galactosidase was purchasedfrom Bethesda Research Laboratories, Bethesda, Md.

Affinity Purification of PLAP using Anti-rPLAP Antibodies

Immunoglobulin enriched fractions of the antisera were prepared byammonium sulfate precipitation. The resulting 1 gG enriched fraction wasnext immobilized on cyanogen bromide activated Sepharose (Pharmacia,Uppsala, Sweden) as suggested by the manufacturer. PLAP was immunopurified as described in Clark, M. A. et al. (1988) Biochem. J. 250:125-132, and Clark, M. A. (1987) J. Biol. Chem. 262: 4402-4406, thedisclosures of both of which are hereby incorporated by reference as iffully set forth herein, except that anti rPLAP antibodies weresubstituted for anti-melittin antibodies. Briefly, whole cell sonicatesmade from CPAE cells were passed over the column which was washedextensively before eluting the bound material with low pH buffer (50 mMsodium acetate pH 3.1). The resulting fractions were assayed for theirability to stimulate PLA₂ activity as described herein.

Synthesis of Single Stranded Antisense DNA and PLAP Peptides

Single stranded DNA was prepared chemically using a Beckman or PharmaciaDNA synthesizer according to instructions for phosphoramidite basedreagents. Synthetic PLAP peptides were prepared using aMilligen/Biosearch (Milford, Mass.) peptide synthesizer according toinstructions for t-BOC amino acids. Purified peptides were prepared bystandard HPLC methods.

Northern Blot Analysis of PLAP mRNA Induction

CPAE or BC3H1 cells were treated with leukotriene D4 (1 μM) for varyingtime periods and total RNA extracted using hot phenol (65° C.).Extracted RNA was electrophoresed on a gel prepared from 1% agarose (10μg/lane) and blotted onto nitrocellulose membrane according to standardprocedures. Probing was performed using a PLAP cDNA clone which had beennick labelled with ³² P dCTP.

Assay for PLAP Neutralizing Activity

PLAP (10 units) purified as described herein was incubated with 2 μl ofammonium sulfate purified anti-rPLAP antibody. The antibody PLAPsolution was added to whole cell sonicates and PLA₂ activity assayed asdescribed herein. Anti-melittin antibodies were found to have noneutralizing activity against PLAP.

Antisense, PLAP, and the Treatment of Rheumatoid Arthritis

Antisense experiments were performed in order to asess the role of PLAPprotein synthesis in the activation of PLA₂ in endothelic cell sinresponse to inflammatory stimuli. It has recently been shown that directaddition of synthetic antisense DNA to cultured cells specificallydisrupts the synthesis of protein [Graham F. I., Vandereb A. J. (1973)Virology 52: 456-463; Heikkila R., Schwap G., Wickson, E. Pluzink D. H.,Watt R., Neekers L. (1987) Nature 328: 445-449]. A single strand ofantisense DNA homologous to the first 20 bases of the 5' end of the openreading frame of the PLAP cDNA clone was synthesized. The antisensesequence had the following sequence: CACTATATGAGCGGCCACTCTAATT (which isthe sequence between the arrows in FIG. 2). This DNA was then added toCPAE bovine endothelial cells at a concentration of 25 mM forapproximately 4 hours prior to the start of the experiment. The additionof antisense to the cell successfully abolished the induction of PLAPprotein synthesis following LTD4 treatment (1 μM) as determined by ELISAassay. PLA₂ activity, arachidonic acid release and prostacyclinsynthesis were also inhibited. These results are summarized in Tables 8,9 and 10. In Table 8, antisense or irrevelant DNA was added to the cellsprior to the measurement of phospholipase A₂ activity as describedherein. All values are expressed as pmol of reaction product producedper min per mg of protein. In Table 9, cells were incubated with ³H-arachidonic acid 10 μCi/ml overnight next antisense or irrelevant DNAwas added to the culture 25 μM for four hours prior to the addition ofLTD. The amount of radiolabeled secreted into the supernatant followingten minutes of treatent was quantitated. All values are expressed as cpmreleased by 10⁵ cells following ten minutes of treatment. In Table 10,cells were incubated with antisense DNA or irrelevant DNA 25 μM for fourhours prior to the addition of LTD₄ (1 μM). The amount of prostacyclinreleased by the cells were quantitated by radioimmunoassays.

From these experiments it was concluded that the mechanism by which LTD4increases prostacylin production in these bovine endothelial cellsrequires that the cells first synthesize PLAP mRNA, an event inhibitableby actinomycin D treatment. Next the PLAP mRNA must be translated inorder for mature PLAP protein to be made. Translation of the PLAPmessage can be inhibited using cycloheximide or antisense PLAP DNA.Irrespective of the method of inhibition it is clear that production ofPLAP is a key step in the release of arachidonic acid followingleukotriene stimulation and that the cellular response to the increasedbiosynthesis following leukotriene stimulation is determined by theability of the cells to selectively express PLAP.

                  TABLE 8                                                         ______________________________________                                        EFFECTS OF ANTISENSE DNA ON LTD.sub.4                                         ACTIVATION OF PLA.sub.2                                                       Treatment       Control  +LTD.sub.4                                           ______________________________________                                        None            7.0 ± 0.5                                                                           22.0 ± 2.0                                        Antisense DNA   6.0 ± 1.0                                                                            9.1 ± 1.1                                        Control DNA     6.0 ± 1.4                                                                           19.0 ± 2.0                                        ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        EFFECTS OF ANTISENSE DNA ON LTD.sub.4 INDUCED                                 RELEASE OF ARACHIDONIC ACID                                                   Treatment        Control  +LTD.sub.4                                          ______________________________________                                        None             4680     11680                                               Antisense DNA    4755      5516                                               Control DNA      4910     11180                                               ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        EFFECTS OF ANTISENSE DNA ON LTD.sub.4 INDUCED                                 PGI.sub.2 SYNTHESIS                                                           Treatment        Control  +LTD.sub.4                                          ______________________________________                                        None             2.4 ± 0.6                                                                           4.3 ± 0.4                                        Antisense DNA    2.2 ± 0.5                                                                           2.1 ± 0.4                                        Control DNA      2.1 ± 0.6                                                                           4.4 ± 0.5                                        ______________________________________                                    

We claim:
 1. A substantially purified nucleic acid sequence coding for afragment of human phosphilipase A₂ activating protein wherein saidfragment is from about six to about thirty amino acids in length andwherein said fragment comprises at least one of amino acids 131, 132,192, 193, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279 and 280 of said phospholipase A₂activating protein, and the remainder of the amino acids of saidfragment are selected from the amino acid sequence of phospholipase A₂activating protein continguous with said at least one amino acid in thedirection of the amino terminus of phospholipase A₂ activating protein,the carboxy terminus of phospholipase A₂, or both.
 2. A substantiallypurified nucleic acid sequence coding for a fragment of humanphospholipase A₂ activating protein selected from the group consistingof ##STR1##
 3. A substantially purified nucleic acid sequence coding fora fragment of human phospholipase A₂ activating protein according toclaim 2 selected from the group consisting of ##STR2##
 4. Asubstantially purified nucleic acid sequence coding for a fragment ofhuman phospholipase A₂ activating protein wherein said fragment is fromabout six to about thirty amino acids in length and wherein saidfragment comprises at least one of amino acids 131, 132, 192, 193, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279 and 280 of said phospholipase A₂ activatingprotein, and the remainder of the amino acids of said fragment areselected from the amino acid sequence of phospholipase A₂ activatingprotein contiguous with said at least one amino acid in the direction ofthe amino terminus of phospholipase A₂ activating protein, the carboxyterminus of phospholipase A₂, or both wherein at least one half of theamino acids of said fragment are selected, in sequence, from amino acids260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279 and 280 of said phospholipase A₂ activatingprotein.